Expanding the repertoire of an ERK2 recruitment site: cysteine footprinting identifies the D-recruitment site as a mediator of Ets-1 binding

Biochemistry. 2007 Aug 14;46(32):9174-86. doi: 10.1021/bi7002058. Epub 2007 Jul 21.

Abstract

Many substrates of ERK2 contain a D-site, a sequence recognized by ERK2 that is used to promote catalysis. Despite lacking a canonical D-site, the substrate Ets-1 is displaced from ERK2 by peptides containing one. This suggests that Ets-1 may contain a novel or cryptic D-site. To investigate this possibility a protein footprinting strategy was developed to elucidate ERK2-ligand interactions. Using this approach, single cysteine reporters were placed in the D-recruitment site (DRS) of ERK2 and the resulting ERK2 proteins subjected to alkylation by iodoacetamide. The ability of residues 1-138 of Ets-1 to protect the cysteines from alkylation was determined. The pattern of protection observed is consistent with Ets-1 occupying a hydrophobic binding site within the DRS of ERK2. Significantly, a peptide derived from the D-site of Elk-1, which is known to bind the DRS, exhibits a similar pattern of cysteine protection. This analysis expands the repertoire of the DRS on ERK2 and suggests that other targeting sequences remain to be identified. Furthermore, cysteine-footprinting is presented as a useful way to interrogate protein-ligand interactions at the resolution of a single amino acid.

Publication types

  • Comparative Study
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alkylation
  • Amino Acid Sequence
  • Animals
  • Binding, Competitive / genetics
  • Catalysis
  • Cysteine / metabolism*
  • Mice
  • Mitogen-Activated Protein Kinase 1 / chemistry*
  • Mitogen-Activated Protein Kinase 1 / genetics
  • Mitogen-Activated Protein Kinase 1 / metabolism*
  • Molecular Sequence Data
  • Phosphorylation
  • Protein Binding / genetics
  • Protein Footprinting*
  • Protein Interaction Mapping
  • Protein Transport / genetics
  • Proto-Oncogene Protein c-ets-1 / chemistry*
  • Proto-Oncogene Protein c-ets-1 / metabolism*
  • Rats
  • Substrate Specificity / genetics

Substances

  • Proto-Oncogene Protein c-ets-1
  • Mitogen-Activated Protein Kinase 1
  • Cysteine